The present invention relates to enzyme recovery and purification and, more particularly, to a process for the recovery of a purified alkaline protease from Bacillus licheniformis and Bacillus alcalophilus, or genetically engineered variants of these, and the composition that results from that process.
The use of enzymes in detergents is well known. Generally, enzymes used for detergent purposes have primarily been the alkaline stable proteases, lipases and alpha-amylases. Of the alkaline proteases, serine proteases derived from Bacillus species, namely Bacillus subtilis, Bacillus licheniformis, and alkalophilic Bacillus bacterial have been widely used in detergent formulations. (Starace C. and Barford, H. C., Encyclopedia Chem. Technol. 9, pp. 138-148 (1980); Koki Horikoshi and Terahiko Akika, A New Microbial World, Springer-Verlag, N.Y., p. 93 (1982)).
Enzymes constitute only a small portion of most liquid detergent formulations. Thus, it is necessary to produce fairly concentrated enzyme preparations. Enzyme concentrates are traditionally prepared by removing the water from aqueous solutions of the enzymes using conventional methods such as ultrafiltration and evaporation.
Inorganic salts such as ammonium sulphate and sodium sulphate have been used extensively to precipitate enzymes from aqueous solution at laboratory and commercial levels. (Dixon, M. and Webb, E. D., Enzymes, Academic Press, N.Y., pp. 39-41 (1964), Curline, Methods of Plasma Protein Fractionation, Academic Press, N.Y. (1980)). The widespread use of these salts on a large scale, however, can pose environmental problems and complicate waste water treatment, in fact, many countries in Europe have already restricted large scale industrial use of these salts. Organic solvents such as ethanol and acetone are also used as precipitants (Dixon and Webb, Enzymes, supra, pp. 37-39; Bauer et al,. J. Chem., 5(3), pp. 117-20 (1967)), however, their use has been limited because of cost and concern for safety.
Environmentally safe and cost effective enzyme purification methods are discussed in, for example, Becker et al., U.S. Pat. No. 5,041,377, describing a method of producing crystalline subtilisin derived from Bacillus subtilis and Bacillus amyloliquifaciens by the addition of a halide salt to an alkaline protease solution at temperatures less than 10.degree. C. and at a pH range from 5.2 to 5.8.
The color and odor of protease can adversely effect the quality of the detergent formulations in which they are incorporated. This necessitates the removal of pigments from the enzyme concentrate, which pigments are believed to be a part of an enzyme-pigment complex. Dixon, M. and Webb, E. C., Enzymes, supra, reported solvent precipitation methods to remove pigment from protease solution. This method, however, resulted in poor product yield. Absorption of pigments with activated carbon from aqueous enzyme concentrate is generally practiced in industrial applications, however, material loss, high cost and waste disposal present major drawbacks.
It is desirable that alkaline protease preparations for detergent applications be free from components which can cause undesirable color, haze, instability and allergic activity in the final product. These components may be derived from the microorganism themselves or from residual fermentation raw materials. In preparations of gram positive Bacilli, cell wall anionic polymers, peptidoglycans and other polysaccharide contaminants become solubilized during cell growth due to cell wall turnover. The presence of these bacterial cell wall polymers in alkaline protease preparations can cause several undesirable effects including an increase in the allergenicity, a decrease in enzyme stability by binding cations, e.g., Ca.sup.++, and may cause haze formation in detergent formulations. Patent Cooperation Treaty ("PCT") Patent Application No. W089/05863 published Jun. 29, 1989 discloses a method to separate these polymers from protease preparations using ion-exchange chromatography. This method is difficult for a large scale operation and expensive. There is a need for a simple and cost-effective method of removing carbohydrate contaminants in general, and galactosyl polymers in particular, from alkaline protease preparations.
Another problem that exists in preparing purified alkaline protease product is a loss of enzymatic activity due to autolytic degradation during either processing or storage. Alkaline proteases perform the function of hydrolyzing protein molecules in the bacterial environment. Their catalytic activity is based on cleavage of the peptide bond in protein molecules and a resultant breakdown of the protein structure into small polypeptides and individual amino acids. Because the alkaline protease is itself a protein molecule, alkaline proteases will begin to hydrolyze other alkaline protease molecules in solution under the proper conditions. While this is a general problem with protease preparations, loss of proteolytic activity as a result of autolytic degradation of alkaline protease may adversely effect the performance of the enzyme under extreme conditions present in detergent formulations, e.g., alkaline pH, because the alkaline protease is most catalytically active at the alkaline pH ranges.
None of the patents, patent applications or publications described above provide the important advantages of an alkaline protease preparation free of haze and odor forming contaminants, allergy causing activity and other undesired fermentation derived impurities. Thus, a need exists for a simple and efficient process for purifying alkaline protease while limiting autolytic degradation of the protease during the purification procedure. The ability to effectively remove allergenic properties and other undesirable characteristics from alkaline protease preparations is critical for effective and safe commercial protease utilization.